Semirational Engineering of the Naphthalene Dioxygenase from Pseudomonas sp. NCIB 9816‐4 towards Selective Asymmetric Dihydroxylation

Abstract

Enzyme-catalyzed asymmetric dihydroxylation is ap owerful tool fort he selective oxyfunctionalization of various organic compounds. By applying Rieske non-heme dioxygenases (ROs), molecular oxygen and ar eduction equivalent are needed for the generation of vicinal cis-diols. We report ac omprehensive mutagenesis study of the active site of the naphthalene dioxygenasef rom Pseudomonas sp. NCIB 9816-4 comprising 62 variants. We aimed to understand the important structure-function relationships by investigating different substituted arene substrates and the geometry of the active site. Introducing single-point mutations at positions F202, A206, V260, H295, F352, and L307 resulted in drastic shifts in the reactions pecificity,r egioselectivity,a nd stereoselectivity (! 90 %) while maintaining the residual activity towards the natural substrate naphthalene.One of the key reactions in chemistryi st he selective oxyfunctionalization of organic compounds. In this context, chiralv icinal 1,2-diols are of particular interest and are often applied as chiral ligands, auxiliaries, and chiral synthons for pharmaceuticals and agrochemicals.[1] These functionalized compounds are generated by multicomponent Rieske non-heme dioxygenases (ROs), which, analogously to some P450sm onooxygenases, consist of ar eductase, af erredoxin, andahexameric oxygenase.[2] In ROs, categorized as types III and IV,t he reductase obtains electrons from the naturalc osubstrate NAD(P)H,a nd a ferredoxin component shuttles electrons from the reductase to the oxygenase active site, at which actual oxygen activation and substrate hydroxylation occur. [3,4] Representing ab iocatalytic alternative to asymmetricd ihydroxylation with toxic transition-metal catalysts, ROs also exhibit ab road substrate scope.[5] Over 300 diverse substrates ranging from polyaromatic hydrocarbons such as phenanthracene to halogenated ethylenes and flavonoid structures were reportedt ob eh ydroxylated with these enzymes. [6,7] Furthermore, ROs were shown to be suitable for bioremediation applications and to have the ability to process xenobiotics. [8,9] In industry, ROs are utilized to manufacture the blue dye indigo, which is crucial for the production of denim cloth.[10]Oxygen-dependent ROsn ot only convert ah uge variety of compounds, but these catalysts also displayabroad reaction scope. With these enzymes ystems it is possible to address monohydroxylations( Scheme 1, path I), dihydroxylations (Scheme 1, path II), and O-dealkylations (Scheme 1, path III) with high regio-a nd stereoselectivities. [11,12] Recently,s emirational engineering of the cumene dioxygenase (CDO) from Pseudomonas fluorescens IP01 expanded the substrate scope of these versatile catalysts to ar ange of sterically demanding cyclic and linear alkenesa nd monoterpenes. The CDOs ingle variant M232Aw as able to convert different terpeneg eometries such as myrcene and (À)-a-pinene with high specificity and selectivity. [13] This work motivated us to gain furtheri nsighti nto crucial structure-function relation...

Biocatalyst Screening with a Twist: Application of Oxygen Sensors Integrated in Microchannels for Screening Whole Cell Biocatalyst Variants

“…The oxygen consumption rate measured in the three oxygen sensors used (2, 3 and 6) is presented in Figure 6 a. Product (1-phenylethanediol) concentration at the different residence times was also measured ( Figure 6 b) in order to compare with previously obtained results [ 44 , 45 ], and allow a correlation between observed oxygen consumption and substrate conversion. As observed, there is formation of the expected product (1-phenylethanediol) in the presence of the NDO and the high conversion in the initial minutes of the reaction agrees with the observed higher oxygen consumption for the wt cells.…”

“…The general protocol followed to obtain the variants/mutants is described in Gally et al (2015) [ 34 ] and further optimized towards a better reproducibility as presented in Halder et al (2017) [ 45 ].…”

“…Hence, styrene was used to compare the different variants in terms of ability to convert this family of substrates. The chosen case study involved the screening of two previously developed dioxygenase variants [ 44 , 45 ] and their comparison with the wild-type NDO.…”

Artificial Light‐Harvesting Complexes Enable Rieske Oxygenase Catalyzed Hydroxylations in Non‐Photosynthetic cells

“…CDO variant M232A converts 1 almost exclusively to 1 a (ee > 98 %), [34] whereas NDO variant H295A shows a different ratio between allylic monohydroxylation and cis-dihydroxylation for several substituted arenes. [35] First, we investigated the expression of the whole RO system under different culture conditions (SDS-PAGE, Figure S2) and confirmed the RO activity by an agar plate assay based on indigo formation (Figures S3 and S4) and could identify significant activity toward indole. [36] To identify the cell density leading to the highest product formation catalyzed by CDO expressed under different culture conditions, biotransformations supplemented with glucose (20 mm) and with 1 (10 mm) as substrate were performed under dark conditions (Table S8).…”

Artifizielle Lichtsammelkomplexe ermöglichen Rieske‐Oxygenase‐ katalysierte Hydroxylierungen in nicht‐photosynthetischen Zellen

“…Die CDO-Variante M232A setzt 1 fast ausschließlich in 1 a um (ee > 98 %), [34] während die NDO-Variante H295A für mehrere substituierte Arene ein unterschiedliches Verhältnis zwischen allylischer Monohydroxylierung und cis-Dihydroxylierung zeigt. [35] Zunächst untersuchten wir die Expression des gesamten RO-Systems unter verschiedenen Kultivierungsbedingungen (SDS-PAGE, Abbildung S2) und bestätigten die RO-Aktivität durch einen Agarplatten-Assay auf der Basis von Indigo-Bildung (Abbildungen S3 und S4) und konnten eine signifikante Aktivität gegenüber Indol feststellen. [36] Um die Zelldichte zu bestimmen, die in der CDO-katalysierten Reaktion zur hçchsten Produktbildung führt, wurden Biotransformationen mit Zellen, die unter verschiedenen Bedingungen kultiviert wurden, im dunklen mit Glukose (20 mm) und 1 (10 mm) durchgeführt (Tabelle S8).…”